One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

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BackgroundExosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients.MethodsExosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4–2 and bone metastatic C4–2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody.ResultsAmong proteins upregulated in C4–2 and C4–2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4–2 and C4–2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues.ConclusionsOur results suggest that serum exosomal GGT activity could be a useful biomarker for PC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3301-x) contains supplementary material, which is available to authorized users.

The authors describe the computed tomography (CT) and magnetic resonance imaging (MRI) findings of an 18-yearold man with renal cell carcinoma (RCC) associated with the Xp11.2 translocation/transcription factor E3 (TFE3) gene fusion (Xp11 translocation carcinoma). The lesion was hyperdense on unenhanced CT, hypovascular on contrast-enhanced studies, hypointense on T2-weighted MR images, and hemosiderin deposition was suspected on phase-shift gradient-echo MR images. Histopathological specimens revealed pathological findings resembling papillary RCC predominantly and exhibited immunoreactivity for TFE3. Because there is often considerable morphological overlap between this carcinoma and papillary RCC, the imaging findings of Xp11 translocation carcinoma may be similar to those of the papillary subtype. Therefore, Xp11 translocation carcinoma should be considered, particularly in young patients when radiologic images demonstrate a renal tumor mimicking the papillary subtype. RENAL CELL CARCINOMA (RCC) associated with Xp11.2 translocation/TFE3 gene fusion (Xp11 translocation carcinoma) was identified %20 years ago and listed as a separate entity in the 2004 World Health Organization (WHO) classification of kidney tumors (1). This RCC subtype is defined by different translocations involving chromosome Xp11.2, which all result in gene fusions involving the TFE3 gene. This carcinoma predominantly affects children and young adults in the second and third decades of life and shows no gender predominance, although some cases have been described in older patients (2-5). Approximately one-third of pediatric RCCs are estimated to be Xp11 translocation carcinomas, whereas the clear cell subtype accounts for 15% of RCCs in children. Compared with pediatric cases, clinical stages in adult cases tend to be more advanced and clinically they usually pursue unfavorable courses (3,5). Liver, lung, and periaortic lymph nodes are common sites of metastasis (3). Although Xp11 translocation carcinomas have been reported from the perspectives of their pathologic and urologic features, we are unaware of any previous reports focusing on their radiological imaging findings. Hence, we describe the computed tomography (CT) and magnetic resonance imaging (MRI) findings of an Xp11 translocation carcinoma in a young adult patient and their relations with pathologic findings. CASE REPORTAn 18-year-old man presented with sudden onset of gross hematuria and abdominal pain in the right flank. On subsequently performed CT images, a mass was found in the anteroinferior segment of the right kidney.CT images were obtained using a 16-slice CT scanner (LightSpeed Ultra 16, GE Healthcare, Milwaukee, WI), and MRI was performed using a 3T MRI system (Achieva Quasar Dual 3T; Philips Medical Systems, Best, the Netherlands). On contrast-enhanced CT and MRI, multiphasic scans were initiated 35 and 180 seconds after initiating contrast injection for the corticomedullary and excretory phases, respectively.On unenhanced CT the mass was a 38 Â 32 Â 41 mm, a well-de...

Mycoplasma genitalium was detected in 21 (14.1%) of 149 vaginal swab samples and in 1 (0.7%) of 149 throat washing samples from female sex workers during 2013–2014 in Japan. Prevalences of M. genitalium with macrolide resistance–associated 23S rRNA mutations and fluoroquinolone resistance–associated parC alterations were 47.1% and 36.8%, respectively.

The authors present a case report of collecting duct carcinoma (CDC) that responded to nivolumab, a programmed death 1 (PD-1) immune-checkpoint-inhibitor antibody, following the failure of systemic treatment with chemotherapy and targeted therapy. The patient underwent right radical nephrectomy and segmentectomy of the lung following chemotherapy. Fifteen months following the first surgery, segmentectomy and subsequent second-line chemotherapy were performed for recurrence in the lung. Targeted therapy with temsirolimus for recurrence of the lung and lymph node metastases was ultimately used for 30 months. However, the temsirolimus treatment failed to suppress the growth of metastatic lesions. Nivolumab resulted in complete response of the lung metastasis, and it stabilized the lymph node metastasis. PD-L1 was highly expressed in both primary tumor and the metastatic regions. Therapy with nivolumab is ongoing. These findings suggest that treatment with nivolumab may be considered for metastatic and treatment-failure CDC.

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Advanced bladder cancer is treated mainly with gemcitabine and cisplatin, but most patients eventually become resistance. Androgen receptor (AR) signaling has been implicated in bladder cancer as well as other types of cancer including prostate cancer. In this study, we investigated the expression and role of AR in gemcitabine-resistant bladder cancer cells and also the potential of enzalutamide, an AR inhibitor, as a therapeutic for the chemoresistance. First of all, we established gemcitabine-resistant T24 cells (T24GR) from T24 bladder cancer cells and performed gene expression profiling. Microarray analysis revealed upregulation of AR expression in T24GR cells compared with T24 cells. AR mRNA and protein expression was confirmed to be increased in T24GR cells, respectively, by quantitative RT-PCR and western blot analysis, which was associated with more potent AR transcriptional activity as measured by luciferase reporter assay. The copy number of AR gene in T24GR cells determined by PCR was twice as many as that of T24 cells. AR silencing by siRNA transfection resulted in inhibition of proliferation of T24GR cells. Cell culture in charcoal-stripped serum and treatment with enzalutamide inhibited growth of T24GR cells, which was accompanied by cell cycle arrest. AR transcriptional activity was found to be reduced in T24GR cells by enzalutamide treatment. Lastly, enzalutamide also inhibited cell proliferation of HTB5 bladder cancer cells that express AR and possess intrinsic resistance to gemcitabine. Our results suggest that enzalutamide may have the potential to treat patients with advanced gemcitabine-resistant bladder cancer with increased AR expression.